Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis

Jeffrey E. Lee, Winnie Luong, David J T Huang, Kenneth A. Cornell, Michael Riscoe, P. Lynne Howell

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5′-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp 197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3′, C2′, and C3′ atoms and by polarizing the 3′-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3′-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg 193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism.

Original languageEnglish (US)
Pages (from-to)11049-11057
Number of pages9
JournalBiochemistry
Volume44
Issue number33
DOIs
StatePublished - Aug 23 2005
Externally publishedYes

Fingerprint

adenosylhomocysteine nucleosidase
N-Glycosyl Hydrolases
Quorum Sensing
Mutagenesis
Biosynthesis
Catalysis
Hydroxyl Radical
Catalytic Domain
Electrons
Salvaging
Methylation
Polyamines
Enzymes
Site-Directed Mutagenesis
Alanine
Methionine
Stabilization
Crystal structure
Atoms
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lee, J. E., Luong, W., Huang, D. J. T., Cornell, K. A., Riscoe, M., & Howell, P. L. (2005). Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis. Biochemistry, 44(33), 11049-11057. https://doi.org/10.1021/bi050493q

Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis. / Lee, Jeffrey E.; Luong, Winnie; Huang, David J T; Cornell, Kenneth A.; Riscoe, Michael; Howell, P. Lynne.

In: Biochemistry, Vol. 44, No. 33, 23.08.2005, p. 11049-11057.

Research output: Contribution to journalArticle

Lee, JE, Luong, W, Huang, DJT, Cornell, KA, Riscoe, M & Howell, PL 2005, 'Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis', Biochemistry, vol. 44, no. 33, pp. 11049-11057. https://doi.org/10.1021/bi050493q
Lee, Jeffrey E. ; Luong, Winnie ; Huang, David J T ; Cornell, Kenneth A. ; Riscoe, Michael ; Howell, P. Lynne. / Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis. In: Biochemistry. 2005 ; Vol. 44, No. 33. pp. 11049-11057.
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